The present invention pertains to polynucleotides derived from staphylococcal genes encoding resistance to streptogramin A or to streptogramin B and chemically related compounds. This invention also relates to the use of the polynucleotides as oligonucleotide primers or probes for detecting Staphylococcal strains that are resistant to streptogramin A or to streptogramin B and related compounds in a biological sample.
In another embodiment, the present invention is directed to the full length coding sequences of the staphylococcal genes encoding for resistance to streptogramin A or to streptogramin B from Staphylococcus and to the polypeptides expressed by these full length coding sequences.
Further, this invention relates to the use of the expressed polypeptides to produce specific monoclonal or polyclonal antibodies that serve as detection means in order to characterize any staphylococcal strain carrying genes encoding resistance to streptogramin A or to streptogramin B.
The present invention is also directed to diagnostic methods for detecting specific strains of Staphylococcus expected to be contained in a biological sample. The diagnostic methods use the oligonucleotide probes and primers as well as the antibodies of the invention.
Streptogramins and related compounds (antibiotics) produced by streptomycetes can be classified as A and B compounds according to their basic primary structures (Cocito, 1979). Compounds of the A group, including streptogramin A (SgA), pristinamycin IIA (PIIA), virginiamycin M, mikamycin A, or synergistin A, are polyunsaturated cyclic macrolactones. Compounds of the B group, including streptogramin B (SgB), pristinamycin B (PIB), virginiamycin S, mikamycin B, and synergistin B, are cyclic peptidic macrolactones (Cocito, 1979). Compounds of both groups, A and B, bind different targets in the peptidyltransferase domain of the 50S ribosomal subunit and inhibit protein elongation at different steps (Aumercier et al., 1992; Di Giambattista et al., 1989).
A decrease in the dissociation constant of PIB is observed in the presence of PIIA because this latter antibiotic provokes a conformational modification of the bacterial ribosome at the binding sites of these molecules. Thus, A and B compounds, which are bacteribstatic when used separately, act synergistically when combined and become bactericidal, mainly against Gram-positive bacteria.
Natural mixtures such as pristinamycin (Pt), synergistin, virginiamycin and mikamycin, are used orally and topically. A semi-synthetic injectable streptogramin, RP59500, consisting of a mixture of derivatives of A and B compounds (Dalfopristin and Quinupristin, respectively) is currently undergoing in vivo experimental and. clinical trials (J. Antimicrob. Agents Chemother. 30 (Suppl. A), entire volume, 1992; Entenza et al., 1995; Fantin et al., 1995; Griswold et, al., 1996; Torralba et al., 1995). Staphylococcal resistance to synergistic mixtures of A and B compounds (Pt MIC≧2 μg/ml) is always associated with resistance to A compounds (PIIA MIC≧8 μg/ml), but not necessarily with resistance to B compounds (Allignet et al., 1996).
To date, four genes encoding resistance to A compounds have been isolated from staphylococcal and enterococcal plasmids. The genes vat (Allignet et al., 1993), vatB (Allignet and El Solh, 1995), and satA (Rende-Fournier et al., 1993) encode related acetyltransferases (50.4-58.3% amino acids), which inactivate streptogramin A and similar compounds. The staphylococcal gene vga (Allignet et al., 1992) encodes an ATP-binding protein probably involved in the active efflux of A compounds. Nevertheless, there continues to exist a need in the art for polynucleotides specific for Staphylococcus resistant to streptogramin A and/or B and related compounds.